乙烯利和1-MCP对菠萝蜜果实中AheAAT和AheERF表达的影响

Effects of Ethephon and 1-MCP on the Expression of AheAAT Gene and AheERF Transcription Factors in Jackfruit Fruit

【目的】研究乙烯(ETH)和1-甲基环丙烯(1-MCP)处理对菠萝蜜果实成熟过程中醇酰基转移酶(AAT)活性、AheAAT和AheERF1/2转录因子表达的影响,为进一步认识菠萝蜜果实AATs在酯类物质合成中的作用及其调控提供理论依据。【方法】以‘海大2号’菠萝蜜果实为试材,果实盛花期选择具有代表性、花期一致的果实挂牌,花后约150 d采收,选择大小和成熟度相同,且无病虫伤的果实,分成3组进行处理。第一组采用1000 mg·L-1 ETH溶液浸泡2—3 min;第二组采用0.5 mg·L-11-MCP熏蒸处理15 h;第三组作为对照组。在果实成熟过程中定期取样,每次取样3—5个果,测定AAT酶活性、AheAAT和AheERF表达等指标。【结果】通过测定AAT酶活性发现,ETH处理促进了果实的成熟衰老,提前了AAT活性高峰出现的时间,但是降低了酶活性。1-MCP处理抑制了果实成熟衰老,在贮藏前期抑制了AAT的活性,延迟了AAT活性高峰出现的时间,并显著降低了AAT的活性。对AheAAT序列分析发现,其全长1380 bp,编码459个氨基酸。AheAAT具有酰基转移酶的保守结构域H-x-x-x-D和L-x-x-YYPLAGR活性位点基序,D(N)F(V)GWG附加基序,属于BAHD醇酰基转移酶家族,其氨基酸序列与苹果和梨的相似性最高。同时,从菠萝蜜果实中克隆获得2个ERF转录因子,分别命名为AheERF1/2,其中AheERF1全长648 bp,编码215个氨基酸,与苹果和菜豆的同源性最高;AheERF2全长657 bp,编码218个氨基酸,与苹果、番木瓜的同源性最高。AheERF1/2的氨基酸序列含有64/65个氨基酸残基组成的AP2/ERF保守序列,具有ERF家族转录因子的特征序列。ETH处理使果实中AheAAT表达高峰提前出现,并且下调了AheAAT的表达。ETH处理对AheERF1/2转录因子的影响与AheAAT相同,并且ETH降低了AheERF1/2转录因子与AheAAT的相关性。1-MCP处理延长了菠萝蜜果实的贮藏期。在1-MCP处理后的贮藏前期,AheAAT和AheERF1/2转录因子的表达呈现显著降低。当AheAAT和AheERF1/2转录因子出现表达高峰时,1-MCP下调了它们的表达。然而,1-MCP对AheERF1/2转录因子与AheAAT的相关性几乎无影响。【结论】ETH处理提前了AAT的活性高峰及AheAAT和AheERF1/2转录因子的表达高峰,降低了AAT的活性,下调了AheAAT和AheERF1/2转录因子的表达,降低了AheAAT与AheERF2转录因子的相关性。1-MCP处理在贮藏前期抑制了AAT的活性及AheAAT和AheERF1/2转录因子的表达,在贮藏后期降低了AAT的活性,下调了AheAAT和AheERF1/2转录因子的表达量。

【Objective】The effects of Ethephon(ETH)and 1-methylcyclopropene(1-MCP)on AAT activities,the expression of AheAAT gene and AheERF1/2 transcription factor were studied during ripening of jackfruit to provide a theoretical basis for further understanding the role of AATS in the synthesis of esters and its regulation.【Method】The test material was jackfruit of Haida2.During the full bloom period,the fruit with the representative flowering period was labeled and harvested about 150 days after flowering.The fruits with the same size and maturity and no disease and insect damage were selected to divide into three groups for treatment.The first group was immersed in 1000 mg·L-1 ETH solution for 2-3 min;the second group was fumigated with 0.5 mg·L-11-MCP for 15 h;the third group,as a control group,was naturally matured at 22℃and 90%relative humidity conditions.During fruit ripening,samples were taken periodically with 3-5 fruits per times.The flesh of the jackfruit was frozen in liquid nitrogen and then stored in an ultra-low temperature refrigerator at-80℃for determination of AAT activities,the expression of AheAAT gene and AheERF1/2 of jackfruit.【Result】By analyzing the activity of AAT enzyme,it was found that ETH treatment promoted the ripening and senescence of jackfruit,advanced the peak of the activity peak of AAT,but reduced AAT enzyme activity.1-MCP treatment inhibited the ripening and senescence of fruits by inhibiting the AAT activity in the pre-storage period,delaying the time of peak AAT activity,and significantly reducing the activity of AAT enzyme.Sequence analysis of AheAAT gene showed that the total length of AheAAT gene was 1380 bp,encoding 459 amino acids.AheAAT had a conserved domain H-x-x-x-D and an L-x-x-YYPLAGR active site motif,a D(N)F(V)GWG add-on motif.The AheAAT gene belonged to the BAHD alcohol acyltransferase family,and its amino acid sequence had the highest similarity to apples and pears.At the same time,two ERF transcription factors,named AheERF1/2,were cloned from the fruit of jackfruit,of which AheERF1 was 648 bp in length,encoding 215 amino acids,and had the highest homology with apple and kidney bean.AheERF2 was 657 bp in length,encoding 218 amino acids,with the highest homology with apples,papaya.The amino acid sequence of AheERF1/2 contained an AP2/ERF conserved sequence consisting of 64/65 amino acid residues and had a characteristic sequence of an ERF family transcription factor.ETH treatment advanced the expression peak of AheAAT gene in jackfruits and down-regulated its expression.The effects of ETH treatment on the expression of AheERF1/2 transcription factor were identical to that of AheAAT gene.ETH treatment decreased the correlation between AheAAT gene and AheERF2 transcription factor.1-MCP treatment prolonged the storage period of jackfruit fruit.1-MCP treatment prolonged the storage period of jackfruit fruit.The expression of AheAAT gene and AheERF1/2 transcription factors were significantly reduced during the pre-storage period after 1-MCP treatment.1-MCP down-regulated the expression levels of AheAAT and AheERF1/2 transcription factor at the peak of their expressions.However,1-MCP had little effect on the correlations of the AheERF1/2 transcription factor with the AheAAT gene.【Conclusion】ETH treatment advanced the peak activity of AAT enzyme,the peak expression of AheAAT and AheERF1/2 transcription factors,down-regulated the expression of AheAAT and AheERF1/2 transcription factors,and decreased the activity of AAT enzyme and the correlation between AheAAT and AheERF2 transcription factors.Compared with the control group,1-MCP treatment inhibited the activity of AAT enzyme,the expression of AheAAT gene and AheERF1/2 transcription factors in the pre-storage phase,decreased the activity of AAT enzyme and down-regulated the expression levels of AheAAT and AheERF1/2 transcription factors during the late storage period.